Journal of Leukocyte Biology Myeloid cells, immune suppression, tumor immunology
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Originally published online as doi:10.1189/jlb.1203604 on May 20, 2005

Published online before print May 20, 2005
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(Journal of Leukocyte Biology. 2005;78:393-400.)
© 2005 by Society for Leukocyte Biology

Detection of the inhibitory neurotransmitter GABA in macrophages by magnetic resonance spectroscopy

D. J. Stuckey*, D. C. Anthony{dagger}, J. P. Lowe*, J. Miller{ddagger}, W. M. Palm*, P. Styles*, V. H. Perry§, A. M. Blamire* and N. R. Sibson*,1

* Experimental Neuroimaging Group, Department of Biochemistry,
{dagger} Experimental Neuropathology Laboratory, Department of Pharmacology, and
{ddagger} Sir William Dunn School of Pathology, University of Oxford, United Kingdom; and
§ CNS Inflammation Group, School of Biological Sciences, University of Southampton, United Kingdom

1 Correspondence: University Laboratory of Physiology, University of Oxford, OX1 3PT, UK. E-mail: Nicola.Sibson{at}physiol.ox.ac.uk


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Macrophages are key components of the inflammatory response to tissue injury, but their activities can exacerbate neuropathology. High-resolution magnetic resonance spectroscopy was used to identify metabolite levels in perchloric acid extracts of cultured cells of the RAW 264.7 murine macrophage line under resting and lipopolysaccharide-activated conditions. Over 25 metabolites were identified including {gamma}-aminobutyric acid (GABA), an inhibitory neurotransmitter not previously reported to be present in macrophages. The presence of GABA was also demonstrated in extracts of human peripheral blood monocyte-derived macrophages. This finding suggests that there may be communication between damaged central nervous system (CNS) tissue and recruited macrophages and resident microglia, which could help orchestrate the immune response. On activation, lactate, glutamine, glutamate, and taurine levels were elevated significantly, and GABA and alanine were reduced significantly. Strong resonances from glutathione, evident in the macrophage two-dimensional 1H spectrum, suggest that this may have potential as a noninvasive marker of macrophages recruited to the CNS, as it is only present at low levels in normal brain. Alternatively, a specific combination of spectroscopic changes, such as lactate, alanine, glutathione, and polyamines, may prove to be the most accurate means of detecting macrophage recruitment to the CNS.

Key Words: inflammation • MRS • mouse • brain


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Inflammatory processes are known to contribute to tissue damage in the central nervous system (CNS) across a broad range of neuropathologies, including Alzheimer’s disease, multiple sclerosis, traumatic brain injury, and ischaemia [1 ]. Although generally beneficial, inflammation can become excessive or inappropriate and lead to tissue destruction [2 ]. Macrophages are a vital component of this inflammatory response and exhibit a variety of functions including phagocytosis of foreign bodies and debris, antigen presentation, and cytokine production [3 ]. Many of these functions may contribute to bystander damage of healthy tissue, and a better understanding of these processes will enable us to combat the detrimental side-effects of inflammation in the CNS.

Magnetic resonance spectroscopy (MRS) provides a noninvasive method to measure the concentrations of tissue metabolites simultaneously and can be performed to equal effect in tissue extracts, cell suspensions, or within the brain in the clinical setting. Protons in a magnetic field can be excited into a higher energy state by applying a short radio frequency (RF) pulse. Over time, the protons relax back to the lower energy state emitting RF waves, which are detected by the MR spectrometer. As the difference in the energy levels (and hence, the frequency of the RF waves emitted) is dependent on the molecular environment of each individual hydrogen nucleus, a given molecule may emit several different frequencies, resulting in a spectrum that is characteristic of the molecule. In addition, the intensity of each line ("resonance") in the spectrum is related directly to the concentration of the molecules in the sample.

Identification of metabolic differences between immune cells and normal brain advances our understanding of macrophage biology and also opens the possibility for the detection and monitoring of inflammatory processes in vivo by MRS techniques. Previously, in vitro MRS studies of macrophages have focused on the lipid components of these cells, and strong lipid resonances have been found in 1H spectra obtained from murine peritoneal macrophages. Similar resonances observed in 1H spectra obtained from patients suffering from a stroke have been suggested to reflect macrophage infiltration of the brain [4 , 5 ]. However, little or no histopathological data, obtained at the same time as the MRS data, are available to support this hypothesis. In experimental stroke, Gasparovic et al. [6 ] reported colocalization at a single time-point between lipid resonances and microglia/macrophages detected histologically. However, in another experimental study of cerebral infarction in rats, an increase in mobile lipids was found to precede macrophage recruitment to the brain [7 ]. Moreover, strong lipid resonances have also been found in 1H spectra obtained from B and T lymphocytes [8 ], human neutrophils [9 , 10 ], C6 tumor cells [11 , 12 ], and apoptotic cells [13 14 15 ], and in multiple sclerosis, lipid resonances are thought to reflect demyelination [16 , 17 ]. Consequently, these lipid resonances cannot be considered to be unique to any one cell type.

In contrast to the lipid components of macrophages, the nonlipid metabolites have received little attention in MRS studies. The metabolism and metabolite content of macrophages has been studied previously in some detail by other methods [18 ]; however, MRS provides a unique method of simultaneously determining concentrations of multiple metabolites. Therefore, in this study, we have used 1H MRS to investigate the nonlipid components of macrophages and to determine changes in metabolite levels on activation. We chose to analyze extracted samples of macrophages instead of live cells, as greater sensitivity could be achieved, enabling the identification of species not previously reported to be present in these cells and yielding more accurate quantitation of changes in metabolite levels on activation. The one-dimensional (1D) 1H spectrum can contain a number of overlapping resonance peaks from molecules with similar chemical groups, which can hamper metabolite assignment and quantitation. Improved visualization of such metabolites can be obtained using 2D techniques such as gradient-selected correlation spectroscopy (gCOSY). Here, interactions between adjacent protons (j-coupling) within each metabolite molecule are exploited to produce new, off-diagonal or "cross" peaks in the 2D plot. These peaks are characteristic of the protons in the individual molecular groups and the presence of different groups within the metabolite, and are therefore more specific than the equivalent 1D spectrum.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell culture
Frozen stocks of RAW 264.7 cells, a murine macrophage cell line [19 ], were resuspended in RPMI-1640 media containing 2 mM glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin and supplemented with 10% heat-inactivated fetal bovine serum [media and supplements from Invitrogen (Paisley, UK); antibiotics from Sigma (Poole, UK)]. Cells were incubated at 37°C in a humidified environment with 5% CO2 and were passaged on alternate days. RAW cells have been used extensively to study the biology of macrophages and display similar functional and adhesion properties to peritoneal macrophages [20 ].

Lipopolysaccharide (LPS) activation
Cells were passaged and then activated with Escherichia coli LPS at a concentration of 50 ng per ml media. The cells were incubated for 24 h at 37°C with 5% CO2 and then harvested for perchloric acid (PCA) extraction. Macrophage activation was confirmed using an interleukin-1ß enzyme-linked immunosorbent assay (R&D Systems, Abingdon, UK) of samples of media from the activated and resting macrophages.

PCA extraction
Culture medium was removed from the flasks, and the cells were rinsed with 0.2% EDTA in phosphate-buffered saline (PBS) to detach them from the flask. Cell viability (determined using Trypan blue exclusion) was 90–95%. Cells (108) were pipetted into separate tubes (n=6 quiescent and n=6 activated samples). All traces of the culture media were removed by three serial washes with PBS, each time followed by centrifugation and removal of the PBS. The cells were then PCA-extracted immediately: HCl (5 ml ice-cold 0.02 M) and 0.7 ml 3 M PCA were added to each cell pellet. The samples were homogenized, left on ice for 30 min, and finally, centrifuged for 10 min at 1500 g. The aqueous layer was removed, neutralized with 10 M potassium hydroxide, and then lyophilized. Before spectroscopic analysis, the lyophilized powder was resuspended in deuterium oxide, pH adjusted to 7.2. Trimethylsilylpropionate (TSP) was added to 1 mM as an internal concentration standard for MRS, and samples were made up to 500 µl with deuterium oxide.

Human peripheral blood monocyte-derived macrophages
For the generation of macrophages, human peripheral blood mononuclear cells were isolated from buffy coats (Bristol Blood Donor Services, UK) by centrifugation over a Ficoll-PaqueTM PLUS (Amersham, Little Chalfont, UK) gradient, according to standard procedures. Monocytes (day 0) were then purified by 90 min adherence on gelatin-coated plates and by extensive washing to remove nonadherent cells. After overnight incubation, monocytes were harvested and then cultured in X-VIVO 10 medium (BioWhittaker, Walkersville, MD) with 1% heat-inactivated autologous serum to allow differentiation into macrophages. On day 6, 1.6 x 108 macrophages were harvested and washed five times in PBS, and the cell pellet was frozen immediately in liquid nitrogen for subsequent PCA extraction as described above.

Nuclear magnetic resonance spectroscopy
High-resolution proton MRS was performed on a 9.4 T vertical bore magnet with a Varian Inova spectrometer (Varian, Palo Alto, CA). Solvent-suppressed WET [21 ] 1D 1H spectra (90° pulse, 1280 transients, 3 s interpulse delay, sweep width of 500 Hz) were acquired in a total acquisition time of 1 h, 4 min, for each sample. Subsequently, 2D 1H-1H gCOSY [22 ] spectra (256 increments, 64 scans per increment, 3 s interpulse delay, sweep width of 5000 Hz in the F1 and F2 dimensions) were acquired in a total acquisition time of ~14 h.

Identification and quantitation of metabolites and statistical analysis
All 1D data were analyzed using WinNMR (Bruker, Coventry, UK). Free induction decays were Fourier-transformed, phase-corrected, and baseline-corrected. Metabolites were identified by comparison of the 1D and 2D spectra to phantom and published data [23 , 24 ]. Potentially ambiguous assignments were confirmed by spiking with the proposed metabolite. All accessible spectral resonances were integrated, and concentrations were calculated relative to TSP. Metabolite concentrations are given in nmol per cell. Significant changes were determined using an independent samples t-test on SPSS (SPSS, Woking, UK).

Preparation of normal mouse brain extracts
To investigate potential in vivo markers of macrophage activity, spectra were compared against extracts of normal brain. C57/B6 mice were killed by dislocation of the neck, and the brains were removed in under 1 min and frozen in liquid nitrogen. The tissue was crushed under liquid nitrogen and PCA-extracted as described above, and 1D and 2D spectra were collected.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Identification of metabolites
Representative 1D 1H spectra of PCA extracts from cultured cells of the RAW 264.7 murine macrophage line under resting and LPS-activated conditions are shown in Figure 1a and 1b , respectively. The corresponding 2D 1H-1H gCOSY from the resting macrophage extract is shown in Figure 2a and clearly aids with peak assignment. A full list of all metabolites identified with their corresponding frequencies is given in Table 1 . It is surprising that a reliable and reproducible resonance (peak 5 in Figs. 1 and 2 ) was observed with MRS features corresponding to GABA. To confirm the assignment of the peak to GABA, a number of methods were used, as discussed below.



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  		Figure 1. Proton spectra from cultured cells of the RAW 264.7 murine macrophage line under (a) resting or (b) LPS-activated conditions and also from wild-type (WT) mouse brain (c). Key: 1, Valine; 2, lactate; 3, alanine; 4, polyamines; 5, {gamma}-aminobutyric acid (GABA); 6, acetate; 7, N-acetyl-aspartyl (NAA); 8, glx; 9, glutamate; 10, succinate; 11, glutamine; 12, glutathione; 13, aspartate; 14, creatine; 15, taurine; 16, glycine.

 


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  		Figure 2. 2D 1H-1H gCOSY spectra from an extract of (a) cultured cells of the RAW 264.7 murine macrophage line under resting conditions and (b) normal mouse brain. Key as Figure 1 plus: 17, B-alanine; 18, asparginine; 19, lysine; 20, threonine; 21, phosphorylcholine; 22, glycophosphorylcholine; 23, phosphorylethanolamine; 24, myo-inositol.

 

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  		Table 1. List of Metabolites Identified in the 1H Spectrum of Cultured Cells of the RAW 264.7 Murine Macrophage Line

 
Confirmation of the presence of GABA
Figure 3a shows an expansion of the GABA region of the 1D spectrum obtained from cultured cells of the RAW 264.7 murine macrophage line under resting conditions. For comparison, the equivalent spectrum from a 10-mM GABA solution is shown in Figure 3b , demonstrating the same resonance frequencies and coupling patterns. The 2D spectrum of the same macrophage extract is shown in Figure 3c and clearly resolves the expected GABA cross peaks at 1.89–2.28 ppm and 1.89–3.01 ppm. Spiking of the macrophage extract with GABA resulted in enhancement of all resonances believed to generate from GABA. Finally, a GABA-specific enzymatic assay, involving the reduction of GABA to succinate via the enzyme GABAase, was performed, and the associated conversion of ß-nicotinamide adenine dinucleotide phosphate (NADP) to reduced NADP measured spectrophotometrically at 340 nm. PCA-extracted samples of cultured cells of the RAW 264.7 murine macrophage line were incubated with GABAase (Sigma), according to the manufacturer’s protocol and compared with standard solutions of GABA. This assay confirmed the presence of GABA in the extracts of resting and LPS-activated macrophages. Nonlinear regression analysis of these data and statistical comparison of the best-fit curves demonstrated that the resting macrophages contained significantly more GABA than the LPS-activated cells (Fig. 4 ; F=187.8, P<0.0001).



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  		Figure 3. Expansion of the GABA region (x-axis common to both spectra) in a 1D 1H spectrum from an extract of cultured cells of the RAW 264.7 murine macrophage line (a) and a (10 mM) GABA phantom (b). The 2D macrophage spectrum (c) identifies the cross peaks (*) that arise from GABA.

 


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  		Figure 4. Enzymatic assay that couples the oxidation of GABA to succinate with the reduction of NADP+, resulting in an optical density change. Assay curves given for 20 mM GABA standard solution ({square}) and cultured cells of the RAW 264.7 murine macrophage line under resting ({blacktriangleup}) and activated (x) conditions. Best-fit curves obtained using nonlinear regression analysis are shown, with r2 values of 0.986, 0.998, and 0.975 for the 20-mM GABA, resting macrophage, and activated macrophage data, respectively. Comparison of these data demonstrated significant differences amongst all three curves: F = 187.8, P < 0.0001.

 
Comparison of activated and resting macrophages
Although 25 metabolites could be identified, accurate quantitation was only possible in a subset as a result of resonance overlap in the 1D spectra. Metabolite concentrations are given in Table 2 . Although 2D spectra positively improve identification, accurate resonance quantitation of such spectra is difficult. Upon activation, there were significant elevations in lactate (P<0.01), glutamine (P<0.001), glutamate (P<0.001), and taurine (P<0.05) and a significant reduction in GABA (P<0.05) and alanine (P<0.001).


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  		Table 2. Comparison of Metabolite Levels in Cultured Cells of the RAW 264.7 Murine Macrophage Line under Resting and LPS-Activated Conditions, Mouse Brain, and Human Brain

 
The cell culture media were analyzed to determine the levels of metabolite release upon activation. Owing to the high concentration of sugars and lactate in the media, obtaining an easily quantifiable spectrum proved difficult. Qualitative analysis of the 1D and 2D spectra suggested that the concentration of GABA and alanine is increased slightly in the media from activated cells. As the level of these metabolites decreases in activated macrophages, these findings suggest that they are excreted from the activated cells rather than metabolized internally.

Identification of potential MRS markers for macrophages
High-resolution extract spectra from resting and LPS-activated macrophages were compared against the spectrum from WT mouse brain (Fig. 1c) . There are clearly many differences between these spectra, the most interesting of which are that the macrophage spectra contain large alanine (3), glycine (16), acetate (6), glutathione (12), and polyamines (4) resonances, which are not conspicuous in the brain extract spectrum. Glycine and acetate are singlets, and the polyamines multiplets at 1.78 and 2.12 ppm and the glutathione multiplet at 2.56 ppm fall in areas where there are few species in the normal brain spectrum. It should be noted that although polyamines can be components of tissue-culture media, in this study, this was not the case, and analysis of the tissue culture media verified that these resonances were not an artifact of tissue culture. Comparison of the 2D spectra from macrophages and normal brain (Fig. 2) demonstrated strong cross peaks arising from the polyamines (1.78–3.06 ppm and 2.12–3.14 ppm) and glutathione (2.99–3.31 ppm). In addition, a cross peak from ß-alanine (2.56–3.18 ppm) was observed in the 2D macrophage spectra that was not detected in extracts of brain tissue.

Human peripheral blood monocyte-derived macrophages
High-resolution extract spectra from human peripheral blood monocyte-derived macrophages exhibited, to a large extent, a similar profile to those obtained from the extracts of cultured murine macrophages (Fig. 5a ). The presence of GABA was clearly evident in the 1D and 2D spectra (Fig. 5b and 5c) and was again confirmed by spiking of the extract with a solution of GABA. Although still clearly present, the polyamine, glutathione, and alanine resonances were less pronounced than in the spectra obtained from cultured murine macrophages.



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  		Figure 5. (a) 1D proton spectrum obtained from an extract of human peripheral blood monocyte-derived macrophages. (b) Expansion of the GABA region of the spectrum shown in (a); *, GABA resonances at 1.89 and 2.28 ppm. (c) 2D 1H-1H gCOSY spectrum from the same extract, in which the cross peaks (*) arising from GABA are clearly evident.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In this study, we have used MRS to investigate the metabolite profiles of cultured cells of the RAW 264.7 murine macrophage line under resting and LPS-activated conditions. Of particular interest is the novel finding of the inhibitory neurotransmitter GABA in these cultured murine macrophages and also in human peripheral blood monocyte-derived macrophages. GABA has not previously been reported to be present in any type of inflammatory cell, and its presence may indicate a means of communication between inflammatory cells and the CNS in pathology. In addition to this, we have demonstrated significant changes in metabolite levels following activation of cultured macrophages, which provide an insight into the mechanisms by which these inflammatory cells act. We have also identified some species in the macrophage proton spectrum that are absent from or give much weaker signals in normal brain extracts. These resonances may provide a noninvasive means of identifying macrophage recruitment to the CNS, particularly with the use of 2D MRS techniques, which largely overcome problems associated with spectral crowding in 1D 1H spectra acquired in vivo.

Identification of GABA in macrophages
The excitatory neurotransmitters glutamate and alanine have previously been reported to be present in immune cells [24 ], although it is unclear whether this implies that immune cells can communicate directly with neurons via these molecules. The presence of a glutamate receptor protein on the macrophage cell surface lends weight to the hypothesis that there is some macrophage-to-neuron communication [25 ]. The excitatory neurotransmitter N-acetyl-aspartyl-glutamate has also been identified in macrophages [26 ] and has been suggested to be involved in intercellular communication. Similarly, the presence of glycine, which can function as a neurotransmitter, has been reported in macrophages [27 ]. Our finding that GABA is also present in macrophages suggests that there may be an intricate mechanism of communication between the cells of the immune system and the brain. Moreover, our studies of the media in which activated and resting macrophages were grown suggest that GABA is released from active cells. The detection of ß-alanine, an agonist of the GABAA receptor [28 ] in macrophages, lends support to this hypothesis. It is interesting that the presence of GABA has not been detected in neutrophils or lymphocytes [29 ].

GABA has been identified in the granule cells of the hippocampus [30 ] and was postulated to play a neuroprotective role by decreasing excitotoxicity. In a similar manner, macrophages may release GABA at the site of inflammation, to which they have been recruited and thus, modulate the excitability of nearby neurons. A further alternative is that GABA is used as an osmolyte and that its reduction upon activation reflects a change in the morphology of activated macrophages [31 ].

Metabolite changes upon activation
Significant increases in lactate, glutamine, glutamate, and taurine were observed on activation of the murine macrophages in addition to significant reductions in GABA and alanine. Macrophages are activated by contact with bacterial endotoxin, which stimulates the immune cell to up-regulate multiple intracellular pathways including cytokine and nitric oxide (NO) production, purine and pyrimidine synthesis, and antigen presentation [3 ]. The metabolic rate of the cells is increased significantly as many more biochemical processes are being performed. Macrophages are 90% glycolytic [32 ], and thus, the large increase in lactate concentration we observed by MRS probably reflects a significant increase in anaerobic metabolism. This increased glucose use may also up-regulate glucogenesis, depleting amino acid precursors and thus, also explaining the observed decrease in alanine [33 ].

One of the most striking findings of this study was that glutamine is not apparent in resting macrophages but is present in significant concentrations following activation. There has been extensive research into the multiple functions of glutamine in immune cells (for review, see Newsholme [34 ]). In macrophages, glutamine is essential for cytokine production, phagocytosis, antigen presentation, NO production, and cell replication (purine and pyrimidine synthesis). Glutamine is also used as a respiratory fuel at a similar rate to glucose in macrophages [35 ]. In addition, glutamate and glutamine provide intermediates for many cellular processes. Thus, the increase upon activation of both of these metabolites may reflect up-regulation of numerous biochemical pathways in the active cell. It is unclear why glutamine is absent from resting macrophages (within the limits of detection for this study), but it is clear that this metabolite is readily synthesized or internalized from the culture medium on activation to support the crucial functions performed by the active macrophage.

Macrophages destroy invading pathogens by synthesizing toxic compounds. However, the macrophage must use methods that enable it to survive these cytotoxic insults themselves. One such strategy is to use taurine to detoxify hypochloric acid by forming the more stable compound taurine chloramine [36 ]. The formation of taurine chloramine not only removes a potentially harmful oxidant from the environment but also can act as an effector molecule that inhibits proinflammatory pathways [37 ]. Thus, the increase in taurine levels on activation may reflect an increased requirement to detoxify hypochloric acid within the cell.

Potential MRS markers of macrophage recruitment
A significant problem that is encountered when moving from in vitro to in vivo 1D 1H MRS is that of increased spectral crowding owing to broader line widths, which makes the identification of spectral markers for any cell type considerably more difficult. In particular, the portion of the 1H spectrum from 2 to 5 ppm, where the vast majority of detectable metabolites appears, is extremely crowded. For this reason, although strong resonances were observed from glutamate, glutamine (activated cells), and glycine in the macrophage spectrum, their usefulness as markers of macrophage recruitment to the CNS in vivo will be limited. Furthermore, glutamate and glutamine are abundant metabolites in the brain. Large resonances from alanine and lactate were also observed in the spectra obtained from extracts of cultured murine macrophages, although the alanine resonance in the human monocyte-derived macrophage spectra was less pronounced. These resonances fall in a less-crowded part of the spectrum (0–2 ppm), and lactate and alanine are only present at low concentrations in normal brain. Lactate has previously been observed by MRS following stroke and correlated with postmortem histology obtained 1 week later, showing massive macrophage infiltration [4 ]. It has also been shown that lactate remains elevated and metabolically active many months after a stroke [38 ], supporting the idea that this may reflect the presence of macrophages. However, lactate elevation can also simply reflect metabolic disruption (e.g., ischaemia) and is not, therefore, entirely specific to macrophage recruitment. Similarly, alanine is not unique to macrophages and has been readily detected in neutrophils [10 ] and lymphocytes [29 ].

2D spectra are less hampered by problems associated with resonance overlap and broad lipid peaks than 1D spectra. Two cross peaks in the 2D spectrum from the cultured murine macrophages were particularly striking: those arising from glutathione and polyamines. Neither glutathione nor polyamine cross peaks were detectable in the mouse brain spectrum, suggesting that they might be useful macrophage markers. Low levels of glutathione (1–3 µmol/g) have recently been detected in human brain in vivo with editing techniques [39 ]. Nevertheless, the presence of glutathione has not been reported in neutrophils or lymphocytes and thus, may prove to be a viable marker for macrophages recruited to the CNS. However, for this to be the case, the cells would have to be present in sufficient numbers (~2x105cells/mm3 from the cultured macrophage data) to yield a detectable change in the background levels of glutathione. Moreover, the glutathione resonances in the spectra obtained from human monocyte-derived macrophages were somewhat smaller. There are few reports of macrophage numbers in human brain following injury or during disease. However, one study in which histopathological data were obtained 22 days after a stroke reported the equivalent of ~1 x 105cells/mm3, and it is unlikely that this was the peak of cell recruitment. Increasing the acquisition time by a factor of four, for example, would halve the number of cells required for detection. Thus, it becomes a question of patient tolerance to the duration of the spectroscopy examination. Recent developments have enabled acquisition of in vivo 2D spectra in a reasonable time-frame [40 ]. Thus, this may prove to be the most successful method of noninvasively monitoring macrophage presence. Although 2D COSY MRS is not routinely available clinically at present, there are no software or hardware restrictions to implementing the sequence on commercial clinical systems as has been demonstrated recently at 1.5 T [41 ] and 3.0 T [42 ].

Although present in neural and non-neural cells, polyamines have not been detected in MRS spectra obtained from human or animal brain. This is largely owing to their low concentrations (<0.5 µmol/g), which are below the detection threshold for in vivo MRS, even when elevated following brain injury [43 ]. However, these metabolites have been detected in lymphocytes and neutrophils [29 ]. Thus, it seems unlikely that polyamines will prove to be a definitive marker for macrophage recruitment to the brain, although they may be a useful marker of general inflammatory activity.

In conclusion, using 1D and 2D 1H MRS techniques, we have identified over 25 metabolites, including the unexpected presence of the inhibitory neurotransmitter GABA in this murine macrophage cell line. GABA was also found in extracts of human monocyte-derived macrophages. This finding, taken with the other evidence discussed, suggests that there may be communication between damaged CNS tissue and recruited macrophages and resident microglia, which could help orchestrate the immune response. Significant changes in lactate, alanine, glutamate, glutamine, taurine, and GABA concentrations were observed between resting and activated cells. The identification of strong resonances from glutathione in the macrophage 2D 1H spectrum, which is only present at low levels in normal brain, may represent a noninvasive marker of macrophage recruitment in vivo, provided background levels of glutathione do not change under pathological conditions. Alternatively, a specific combination of spectroscopic changes, such as lactate, alanine, glutathione, and polyamines, may prove to be the most accurate means of detecting macrophage recruitment.


   ACKNOWLEDGEMENTS
 
This work was supported by the Medical Research Council (MRC) under the MRC-funded cooperative, entitled The Host Response to Acute Brain Injury (D. C. A., P. S., V. H. P., and N. R. S.) and Program Grant G8307179 (J. M.).

Received December 1, 2003; accepted April 22, 2005.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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